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Clinical Chemistry 54: 665-672, 2008. First published February 15, 2008; 10.1373/clinchem.2007.100511
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Right arrow Automation and Analytical Techniques
(Clinical Chemistry. 2008;54:665-672.)
© 2008 American Association for Clinical Chemistry, Inc.

Automation and Analytical Techniques

Measurement of Folate in Fresh and Archival Serum Samples as p-Aminobenzoylglutamate Equivalents

Rita Hannisdal1,2,a, Asbjørn Svardal1,2 and Per Magne Ueland1,2,3

1 LOCUS for Homocysteine and Related Vitamins and 2 Section for Pharmacology, Institute of Medicine, University of Bergen; 3 Haukeland University Hospital, Bergen, Norway.

aAddress correspondence to this author at: Section for Pharmacology, Institute of Medicine, University of Bergen, N-5021 Bergen, Norway. Fax 47-55-97-46-05; e-mail rita.hannisdal{at}farm.uib.no.

Background: The development of accurate and precise folate assays has been difficult, mainly because of folate instability. Large interassay and interlaboratory differences have been reported. We therefore developed a serum folate assay that measures folate and putative degradation products as p-aminobenzoylglutamate (pABG) equivalents following oxidation and acid hydrolysis.

Methods: Serum was deproteinized with acid in the presence of 2 internal calibrators ([13C2]pABG and [13C5]5-methyltetrahydrofolate). 5-Methyltetrahydrofolate and other folate species in serum were converted to pABG by oxidation and mild acid hydrolysis. pABG and its internal calibrators were quantified by liquid chromatography–tandem mass spectrometry (LC-MS/MS).

Results: The limit of quantification was 0.25 nmol/L, and the assay was linear in the range 0.25–96 nmol/L, which includes the 99.75 percentile for serum folate concentrations in healthy blood donors. Within- and between-day imprecision was ≤5%. We detected no residual folate in serum samples after sample preparation. Folate concentrations in fresh serum samples obtained with the pABG assay and with a microbiologic assay showed good agreement (r = 0.96). In stored samples containing low folate concentrations due to folate degradation, the pABG assay yielded substantially higher folate concentrations than the microbiologic assay.

Conclusions: The pABG assay combines automated sample preparation with LC-MS/MS analysis. It allows measurement of folate not only in fresh samples of serum/plasma but also in stored samples in which the folate has become oxidized and degraded to an extent that it cannot be assayed with traditional folate assays.




The following articles in journals at HighWire Press have cited this article:


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Analytical Recovery of Folate Degradation Products Formed in Human Serum and Plasma at Room Temperature
J. Nutr., July 1, 2009; 139(7): 1415 - 1418.
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R. Hannisdal, P. M. Ueland, and A. Svardal
Liquid Chromatography-Tandem Mass Spectrometry Analysis of Folate and Folate Catabolites in Human Serum
Clin. Chem., June 1, 2009; 55(6): 1147 - 1154.
[Abstract] [Full Text] [PDF]

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Cancer Epidemiol. Biomarkers Prev., October 1, 2008; 17(10): 2625 - 2631.
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